Autophagic flux is impaired in the brain tissue of Tay-Sachs disease mouse model

Tay-Sachs disease is a lethal lysosomal storage disorder caused by mutations in the HexA gene encoding the α subunit of the lysosomal β-hexosaminidase enzyme (HEXA). Abnormal GM2 ganglioside accumulation causes progressive deterioration in the central nervous system in Tay-Sachs patients. Hexa-/- mouse model failed to display abnormal phenotype. Recently, our group generated Hexa-/-Neu3-/- mouse showed severe neuropathological indications similar to Tay-Sachs patients. Despite excessive GM2 ganglioside accumulation in the brain and visceral organs, the regulation of autophagy has not been clarified yet in the Tay-Sachs disease mouse model. Therefore, we investigated distinct steps of autophagic flux using markers including LC3 and p62 in four different brain regions from the Hexa-/-Neu3-/- mice model of Tay-Sachs disease. Our data revealed accumulated autophagosomes and autophagolysosomes indicating impairment in autophagic flux in the brain. We suggest that autophagy might be a new therapeutic target for the treatment of devastating Tay-Sachs disease.


Introduction
Tay-Sachs disease (TSD) is one of the lysosomal storage diseases (LSD) caused by a mutation in the HexA gene, encoding for the α subunit of lysosomal β-hexosaminidase A (HEXA), responsible for the degradation of GM2 to GM3 ganglioside [1]. Consequently, in TSD pathology, accumulated GM2 in neurons leads to neuronal death and progressive neurodegeneration in affected children. Tay-Sachs patients also have developmental delay, muscle weakness, spasticity, dementia, blindness, and epilepsy followed by death at the age of two to four. Hexa-/mice were generated for further thought of TSD's pathophysiology [2,3]. However, Hexa-/mice did not show any neurological phenotype although the presence of limited GM2 ganglioside accumulation in neurons. Recently, we generated a mice model with a combined deficiency of Hexa and Neu3 genes which showed abnormalities in the size and numbers of lysosomes in all tissues studied, especially in the brain due to abnormal GM2 accumulation. Hexa-/-Neu3-/-undergo progressive neurodegeneration with neural loss and Purkinje cell depletion and survived up to 5 months. In addition, neuroinflammation has been postulated as a pathophysiological mechanism due to the excessive activation of glial cells and the infiltration of numerous inflammatory cells in the brain of Hexa-/-Neu3-/-mouse [4,5].
In mammalian cells, two major mechanisms are carried out for the degradation of intracellular proteins: the ubiquitin-proteasome system (UPS) and autophagy [6]. In particular, autophagy is involved in lysosome-dependent pathways for damaged organelles, unfolded proteins, and accumulated cellular components to maintain cellular homeostasis [7]. Autophagic flux includes vesicle trafficking and a network in which newly produced autophagosomes (doublemembrane vesicles) are fused with lysosomes to degrade autophagic cargo. In this process, autophagore formation, autophagosome completion with the closure of the membrane, and autophagosome-lysosome fusion (autolysosome) take place respectively [8]. Each step of autophagic flux is finely regulated by specific protein complexes and the initiation step, the sequestering of autophagic cargo within an isolation membrane (phagophore), is controlled by the Beclin-1 [9]. Autophagy-related (Atg) proteins generate phagophore assembly sites and enable the envelopment of cytoplasmic material. In particular, the Atg9 protein is a key regulator of autophagy induction. During the maturation of autophagosome, Atg7 has involved in the conversion of the cytosolic form of microtubule-associated protein 1 light chain 3 (LC3-I) to LC3-II which is located on both inner and outer autophagosomal membranes [9]. LC3 involves in phagophore edge folding resulting in autophagosome formation. After the formation of autolysosomes formed by the combination of autophagosomes and lysosomes, LC3-II on the outer membrane is converted back to LC3-I and then intra-autophagosomal LC3-II is degraded by lysosomal hydrolyses. Therefore, the level of LC3-II as a marker of dynamic autophagosomal membranes is generally studied to monitor autophagic activity [10]. In addition, the level of ubiquitin-binding scaffold protein p62 (aggregated endogenous substrates) which know to be associated with LC3-II in the autophagosome is mostly evaluated as a marker of termination of autophagy [9,11]. A bunch of studies also showed a defect in autophagic flux and secondary accumulation of autophagic substrates such as autophagosomes in several LSDs [6,[12][13][14].
Gangliosides, known as glycosphingolipids in humans, that are mainly found in membranes of neurons contribute to promoting axon-myelin interactions, activation of transmembrane receptor signaling, and Ca 2 + homeostasis [15][16][17][18][19]. However, the precise molecular mechanisms underlying their physiological or pathological activities are poorly understood. It has been shown that gangliosides released under pathological conditions may induce autophagic cell death of astrocytes in vitro [20]. In addition, Matarrese et al. have shown that GD3 ganglioside actively contributes to the biogenesis and maturation of autophagic vacuoles upon induction [21]. Activation of autophagy-dependent α-Syn clearance using GM1 ganglioside was also demonstrated in experimental models of Parkinson's disease (PD) in vivo and in vitro [22]. GM2 ganglioside, on the other hand, is an intermediate substrate for biosynthesis and degradation of complex brain gangliosides such as GM1a, GD1a, GD1b, and GT1b [23]. Therefore, the effect of abnormally accumulated GM2 ganglioside in neurons is important for cellular processes including autophagy flux. In the current work, we examined whether accumulated GM2 ganglioside in lysosomes causes alteration in autophagic machinery in four brain regions of early-onset TSD mice model by qRT-PCR, Western Blot, and immunohistochemical techniques. Dysfunctional autophagy was demonstrated in Hexa-/-Neu3-/-mice as indicated by the increase in LC3-II and accumulation of autophagosomes. Our results provide a guide for future work that elucidates the contribution of altered autophagy to TSD pathology.

qRT-PCR analysis
Total RNA was extracted from the cortex, cerebellum, thalamus, and hippocampus from 2and 5-month-old WT, Hexa-/-, Neu3-/-, Hexa-/-Neu3-/-mice (n = 3) using Trizol Reagent (GeneAid) and cDNA was synthesized using reverse transcription kit (Applied Biosystems) following manufacturer's instructions. Relative mRNA expression analysis of the following autophagy-related genes, Beclin-1, Atg9, Atg7, and p62, were analyzed by Roche LightCycler 96 machine using Real-Time SYBER green PCR master mix (Roche, Swiss) with these conditions: initial denaturation at 95˚C for 10 minutes; 45 cycles at 95˚C for 20 seconds and 60˚C for 15 seconds. GAPDH gene expression was used as an endogenous control. The primers used for expression analysis are listed in Table 1.

Discussion
Lysosomal dysfunction resulting from the accumulation of endogenous substrates in lysosomes is the hallmark of LSDs including GM2 gangliosidosis [24]. A bunch of studies on LSDs reported not only lysosomal storage but also impairment of other cellular processes such as calcium homeostasis, lipid synthesis, and signaling pathways [24][25][26]. Owing to the essential role of lysosomes in autophagy, it was reasonable to expect that accumulated GM2 ganglioside in lysosomes of Hexa-/-Neu3-/-mice could have an effect on autophagic flux. However, the relation between accumulated GM2 ganglioside and the autophagy process has not been identified. In the present study, we investigated whether autophagic flux is altered in the brain of Hexa-/-Neu3-/-mice correlated with insufficient lysosomal due to accumulation of GM2 ganglioside.
Previously it was shown that the level of autophagy substrates, such as p62/SQSTM1 are significantly increased in several LDS indicating an impairment of the autophagic flux. p62/ SQSTM1 protein, a component of ubiquitinated protein aggregates, is responsible for targeting polyubiquitinated proteins to autophagosomes [29] and degraded in the termination of the autophagic pathway along with autophagic cargo [30]. Accumulation of p62/SQSTM1 has been reported in the endosomal/lysosomal fraction of npc1-/-mouse brain lysates [31], cultured fibroblasts of Fabry patients [32], mucolipidosis type II, III [33] and type IV (MLIV) fibroblasts [34], muscle fibers of Pompe disease [35], and brain of Gaucher disease mice model [36]. Similarly, in our study, we showed accumulated p62/SQSTM1 in the brain of 5-monthold Hexa-/-Neu3-/-mice suggesting the impairment in the termination step of autophagic flux.
Our knowledge of molecular and cellular mechanisms for Tay-Sachs disease is mostly limited to what we have learned from skin fibroblast and iPSCs obtained from patients with Tay-Sachs disease and recently generated mice model with combined deficiency of β-hexosaminidase A and neuraminidase 3. To our knowledge, this is the first study demonstrating progressive alterations in the autophagic flux in the brain tissue of mice with Tay-Sachs disease. Our findings provide insights into the dysregulation of autophagy in the brain and suggest a potential therapeutic approach to reduce lysosomal accumulation by targeting the regulation and activation of proper autophagy. However, further in vitro and in vivo studies are necessary to elucidate the precise molecular mechanisms underlying dysregulated autophagy in neurons and glial cells of the Tay-Sachs mice model.